Quantitative assays involving the binding of a given test analyte in a sample to a reagent present on a solid support make use of calibrators with which the samples tested in the assays are compared. In some assay systems, such as the radio-immunoassay (RIA) or enzyme-labelled immunosorbent assay (ELISA), where analyses of many samples are conducted simultaneously, it is ususal and convenient to include appropriate calibrators. However, when analyses are made of one sample at a time, which is typical of "doctor's office" assays, the inclusion of a set of calibrators for each sample will make the test more difficult and expensive to carry out. The results from the assays of such samples are therefore most often compared to the results obtained with a set of previously generated calibrators which is used over and over again.
Such externally calibrated assays cannot take account of variations in assay parameters such as incubation time, the temperature at which the assay is conducted, the liquid volumes of the sample and reagents and the concentration of reagents.
In an attempt to remedy the drawbacks inherent in using external calibration in an assay, EP 253 464 discloses a method of providing internal calibration in an assay in which a receptor for an analyte in a sample and a receptor for a conjugate of an analyte receptor with a label substance are immobilized in two separate areas on a solid support. The solid support is contacted with a sample containing the analyte to bind any analyte in the sample to the analyte receptor, after which the solid support is contacted with the conjugate which binds to the analyte in the sample as well as to the receptor for the conjugate.
Although the internal calibration provided in this assay takes several of the possible variations influencing signal development into account, the assay still has to be performed carefully with respect to the volume of the sample added, which means that the system remains relatively sensitive to outside influence. Thus, if a double volume of the sample is erroneously added, the concentration of analyte determined for the sample will be the double of the actual concentration.
The object of the present invention is to provide an assay method based on internal calibration for determining the concentration of a test analyte in a sample, which method does not show variations with respect to the above-mentioned critical parameter.